Reduced dynamic loads due to hip dislocation induce acetabular cartilage degeneration by IL-6 and MMP3 via the STAT3/periostin/NF-κB axis

Developmental dysplasia of the hip (DDH) is characterized by anatomical abnormalities of the hip joint, ranging from mild acetabular dysplasia to hip subluxation and eventually dislocation. The mechanism underlying the cartilage degeneration of the hip joints exposed to reduced dynamic loads due to hip dislocation remains unknown. We established a rodent hip dislocation (disarticulation; DA) model of DDH (DA-DDH rats and mice) by swaddling. Expression levels of periostin (Postn) and catabolic factors, such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (Mmp3), increased and those of chondrogenic markers decreased in the acetabular cartilage of the DA-DDH models. Postn induced IL-6 and Mmp3 expression in chondrocytes through integrin αVβ3, focal adhesion kinase, Src, and nuclear factor-κB (NF-κB) signaling. The microgravity environment created by a random positioning machine induced Postn expression in chondrocytes through signal transducer and activator of transcription 3 (STAT3) signaling. IL-6 stimulated Postn expression via STAT3 signaling. Furthermore, cartilage degeneration was suppressed in the acetabulum of Postn−/− DA-DDH mice compared with that in the acetabulum of wild type DA-DDH mice. In summary, reduced dynamic loads due to hip dislocation induced acetabular cartilage degeneration via IL-6 and MMP3 through STAT3/periostin/NF-κB signaling in the rodent DA-DDH models.


Results
Cartilage degeneration in the acetabulum of DA-DDH rats. We successfully generated a DA-DDH rat model ( Supplementary Fig. 1a). Body weight of the DA-DDH rats was approximately 10% lower than that of the control rats ( Supplementary Fig. 1b). The radiograms revealed hip dislocation and the hypoplastic acetabulum in the DA-DDH rats (Fig. 1a). Indeed, macroscopic assessment of the 4-week-old DA-DDH rats showed that the acetabulum cavity was shallow and the femoral head was small and oval shaped, compared with those in the control rats (Fig. 1b). Histologically, in the 10-day-old DA-DDH rats, the femoral head was dislocated; however, safranin O (SO) staining in the acetabular surface was not decreased, compared with that in the control rats ( Supplementary Fig. 2a). In the 4-week-old DA-DDH rats, the acetabular surface and femoral head were not in contact, the joint cavity was filled with fibrous tissue, and SO staining in the acetabular surface was less intense than that in the control rats (Fig. 1c). In both 10-day-and 4-week-old DA-DDH rats, the anterior wall of the acetabulum had atrophied.
The expression of type II collagen α1 (Col2a1), aggrecan (Acan), type X collagen α1 (Col10a1), matrix metalloproteinase 13 (Mmp13), and type I collagen α1 (Col1a1) in the acetabular cartilage of the 10-day-old DA-DDH rats was comparable to that in the control rats ( Supplementary Fig. 2b). The expression of Col2a1, Acan, and Col10a1 was downregulated, but the expression of Mmp13 and Col1a1 did not significantly change in the 4-week-old DA-DDH rats compared to that in the control rats (Fig. 1d). In situ hybridization analyses showed no differences in Col2a1 and Col1a1 expression between the 10-day-old control and DA-DDH rats (Supplementary Fig. 2c) but revealed downregulated expression of Col2a1 and upregulated expression of Col1a1 in the acetabular cartilage of the 4-week-old DA-DDH rats (Fig. 1e). Second-harmonic generation (SHG) signal of the acetabular superficial cartilage was weaker in the 10-day-old DA-DDH rat than in the control rat ( Supplementary Fig. 2d). The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunostaining for Ki67 showed that apoptosis and cell proliferation in the acetabular cartilage of the 10-day and 4-week-old DA-DDH rats were comparable to those in the control rats ( Supplementary Fig. 2e,f).
Total RNA-sequencing (RNA-seq) analysis of the acetabular cartilage of the DA-DDH rats. Of genes whose expression levels differed between the 3-week-old control and DA-DDH rats by more than twofold, 81 genes were significantly differentially expressed between the two groups (P < 0.05). Among these, the expression levels of 21 genes were upregulated, where those of 60 genes were downregulated. The mechano-sensitive gene, Prg4, was among the 60 genes with downregulated expression (Fig. 2a). Gene Ontology (GO) analyses of these 81 genes showed functional annotation clustering of keywords such as "extracellular region, " "proteinaceous extracellular matrix (ECM), " "ECM, " and "extracellular region part" (Fig. 2b). Cell adhesion-related and biological adhesion-related genes were enriched among the 21 genes whose expression levels were upregulated in the DA-DDH rats. In contrast, cartilage development and chondrocyte differentiation-related genes were enriched among the 60 genes whose expression levels were downregulated in the DA-DDH rats (Fig. 2c). Three cell adhesion-related genes with upregulated expression and four chondrogenesis-related genes with downregulated expression were detected among the ECM-related genes in DA-DDH rats (Fig. 2d).
Postn expression induced by dynamic load reduction is mediated via STAT3 signaling. We cultured rat primary chondrocytes under a virtual microgravity (MG) state using an RPM ( Supplementary Fig. 4a).
The expression levels of Postn in the chondrocytes cultured under MG were upregulated, compared with that in the chondrocytes cultured under 1G condition (Fig. 5a,b). The expression levels of IL-6 and Mmp3 were upregu- www.nature.com/scientificreports/  www.nature.com/scientificreports/ lated, that of Col2a1 was downregulated, and those of Acan and Col1a1 remained unchanged in the chondrocytes cultured in MG (Fig. 5a). The expression of phosphorylated STAT3 (p-STAT3) was higher in chondrocytes cultured in MG for 3 h than in chondrocytes cultured under 1G condition, but the expression of p-p65, p-p38, p-Erk1/2, and β-catenin remained unchanged ( Fig. 5c and Supplementary Fig. 4b). The expression of p-STAT3 and p-p65 was higher in chondrocytes cultured in MG for 24 h than under 1G condition (Fig. 5c). Furthermore, the increased expression of Postn in chondrocytes cultured under MG was suppressed in chondrocytes treated with Stattic, an inhibitor of STAT3 signaling. However, a similar suppression was not observed in cells treated with BAY11-7082 (Fig. 5d). Cartilage degeneration is suppressed in Postn −/− DA-DDH mice. We generated the DA-DDH mouse model ( Supplementary Fig. 6a). Although the hip joint of 8-week-old DA-DDH mice was not completely dislocated, the acetabulum and the femoral head had poor compatibility, the anterior and inferior walls of acetabulum were hypoplasia, the posterior wall was thickened, and the femoral head was flattening (Supplementary Fig. 6b,c). These phenotypes were similar with the rat DA-DDH model. In Postn −/− DA-DDH mice, cartilage degeneration in the anterior wall of the acetabulum was markedly suppressed relative to that in WT DA-DDH mice ( Fig. 7a and Supplementary Fig. 7a). Additionally, the expression of Col2a1 was downregulated, whereas the expression of Col1a1 and Postn was upregulated, in the acetabular cartilage of the WT DA-DDH mice ( Fig. 7a and Supplementary Fig. 7b). The downregulation of Col2a1 expression and upregulation of Col1a1 expression were suppressed in the acetabular cartilage of the Postn −/− DA-DDH mice. Furthermore, the expression of Postn, p-p65, IL-6, and Mmp3 was upregulated in the acetabular cartilage of the WT DA-DDH mice, but this upregulated expression was suppressed in the Postn −/− DA-DDH mice (Fig. 7a,b and Supplementary  Fig. 7c). Immunostaining revealed that p-STAT3 expression was increased in the acetabular cartilage of the WT DA-DDH mice but suppressed in the Postn −/− DA-DDH mice ( Fig. 7c and Supplementary Fig. 7d), indicating that downstream targets of Postn could stimulate STAT3 signaling. These results showed that the loss of Postn expression protected against cartilage degeneration in the acetabulum of DA-DDH models by suppressing the expression of IL-6 and Mmp3 via NF-κB signaling. Thus, we demonstrated that dynamic load reduction in the dislocated hip associated with DDH induced acetabular cartilage degeneration by activating the STAT3/Postn/ NF-κB/IL-6 and Mmp3 feedback loop (Fig. 8).

Discussion
This study focused on the mechanism by which cartilage degeneration is induced in articular cartilage exposed to reduced dynamic load. Prg4 expression was downregulated in the acetabular cartilage of DA-DDH rats. The anterior wall of the acetabulum in DA-DDH models was atrophied. SHG can evaluate collagen orientation in unstained tissue sections 20 . Moreover, it can identify changes in collagen orientation before changes in gene expression in tissues take place. Although SHG signaling intensity in cartilage subjected to excessive stress or traumatic damage is stronger than that under normal conditions 20 , in our study, the SHG signal of the cartilage of the anterior wall of the acetabulum in the DA-DDH rat was weaker than that in the control rat. Furthermore, wear, fibrillation and lack of hyaline cartilage observed in traumatic knee OA models such as destabilization of the medial meniscus model were not observed in the acetabular cartilage of DA-DDH models, but a fibrous change of hyaline cartilage was observed in their acetabular cartilage. Based on these results, we inferred that dynamic load on the acetabular cartilage in the dislocated hip of DA-DDH rats, especially on the cartilage of the atrophied anterior wall, was reduced. Therefore, we suggest that mechanisms different from excessive stress or traumatic damage-induced OA were involved in cartilage degeneration in the dislocated hip of DDH. Postn is expressed in tissue exposed to load, as well as in damaged tissue, and promotes cell proliferation and migration, dedifferentiation, and tissue degeneration and reconstruction by activating several signaling pathways 13,16,17,21,22 . Postn interacts with other ECM molecules and induces fibrous tissue growth to contribute to   We also revealed that the expression of Postn, IL-6, and Mmp3 was upregulated and that of Col2a1 and Acan was downregulated in the articular cartilage of the unloaded acetabulum in DA-DDH models, compared with that in controls. In addition, Postn induced the expression of IL-6 and Mmp3 via integrin-FAK-Src-NF-κB signaling. We also showed that Postn suppressed the expression of Col2a1 and Acan in chondrocytes. IL-6 is a pro-inflammatory cytokine and induces the degeneration of various ECM components [28][29][30][31] . MMP3 is a type of stromelysin that plays an essential role in tissue remodeling by degenerating ECM 32 and inducing the breakdown of proteoglycans in the cartilage in OA and rheumatoid arthritis 33,34 . The expression of MMP3 is reportedly upregulated in the articular cartilage of DDH-related hip OA patients 35 . We showed that the expression levels To reproduce in vivo conditions in an in vitro culture system such that the chondrocytes were not stimulated with sufficient mechanical load, we cultured chondrocytes under a virtual MG state using an RPM. We observed that dynamic load reduction induced Postn expression via STAT3 signaling. In addition, the expression of Col2a1 was downregulated in chondrocytes cultured under the MG condition. Moreover, IL-6, a downstream target of Postn, upregulated Postn expression via STAT3 signaling. Our findings provide supporting evidence that reduced dynamic load can promote acetabular cartilage degeneration by activating the STAT3/Postn/NF-κB/ IL-6 and Mmp3 feedback loop in an autocrine or paracrine manner and may suppress chondrogenesis during the hip dislocation of DDH.
This study has a few limitations. We did not consider genetic [36][37][38][39][40] or embryonic factors. DA-DDH rats showed approximately 10% lower body weight than the control rats. In DA-DDH models, nutritional deficiency, inflammation, tissue contusion, ischemia, wear and tears around the joint caused by swaddling may have affected cartilage homeostasis. Additionally, loss of synovial fluid might be biologically harmful to acetabular chondrocytes due to the loss of nutrition and synovial fluid derived stem cells, which are important to cartilage repair. The www.nature.com/scientificreports/ impaired perichondrium, a chondrocyte progenitor cell niche, might also be involved. Furthermore, our in vivo results suggest that Postn expression in the acetabular cartilage might be induced by the paracrine effect of Postn derived from fibrous tissue in and around the hip joint in DA-DDH models. Therefore, in future studies, Postn expression should be disrupted in a temporally controlled and cartilage-specific manner by genetic ablation in postnatal mice to investigate the acetabular cartilage after hip dislocation. Acetabular hypoplasia and hip dislocation occur to varying degrees during the natural process of DDH. However, we did not examine the degree and incidence of hip dislocation and acetabular dysplasia of rats with swaddling in detail. The mechanism of cartilage degeneration in mild hip dysplasia may differ from that of complete hip dislocation. In DA-DDH mice, although the hip joint did not completely dislocate, subluxation between the acetabulum and the femoral head caused similar changes with DA-DDH rats. Postn −/− mice exhibit some skeletal abnormalities, which complicate mechanistic exploration in cartilage 14 . In the future, acetabular cartilage with DDH of various degrees should be investigated and compared to non-DDH to validate the underlying mechanism.
In conclusion, we demonstrated that dynamic load reduction in the dislocated hip associated with DDH induced acetabular cartilage degeneration by activating the STAT3/Postn/NF-κB/IL-6 and Mmp3 feedback loop in an autocrine or paracrine manner. We showed one pathway of mechanical-biological processes of chondrocytes in cartilage degeneration associated with DDH, but further studies are needed for understanding complex biological processes involving many cell types in DDH. www.nature.com/scientificreports/

Methods
Animals. All methods were performed in accordance with the relevant guidelines and regulations. All experimental protocols were approved by the Gifu University Animal Experiment Committee (H30-099). The animal experiments were conducted in compliance with the Animal Research: Reporting in vivo experimental guidelines. Information on the animals used has been provided in Supplementary Table 1. Postn −/− mice were provided by the Material Management Center in Kyushu University 13 . We crossed these mice and C57BL/6J mice to generate the Postn −/− and Postn +/+ littermate controls. Rats and mice were housed in individually ventilated cages in a hygienic barrier facility operating at 23 ± 1 °C and 50 ± 10% humidity. The controls and DA-DDH models used for in vivo analyses were littermate and housed in the same cages. Food and water were available freely, and photoperiod lasted from 8:00 to 20:00.
Establishment of animal DA-DDH models. The DA-DDH rats were generated as described previously 11,12,41,42 . Since the lower extremities of the neonate mice were too short for swaddling, to establish the DA-DDH mice, we threaded 5-0 prolene (Ethicon) onto the distal femur and proximal tibia of two-weekold C57BL/6 mice, tied them, and swaddled in hip adduction and knee extension positions for two weeks. The surgical tape for swaddling was changed in the DA-DDH models once every two days, and they were released from the fixing for several hours at that time. Water bottles and food were placed on the floor of the cages. More than three rats or mice were used in each group to obtain unbiased and reliable results; the sample size for each experiment is indicated in the figures representing the corresponding results. Rats and mice were divided into DA-DDH and control groups by randomization; sex was not evaluated. The body weight of the rats was evaluated over time. A few rats and mice had to be excluded from the evaluation owing to mortality due to gastrointestinal disorders induced by abdominal compression, but no other exclusion criteria were set. Flow charts for the rat and mouse in vivo experiments were showed in Supplementary Figs. 1a and 5a. The numbers of animals used in this study are summarized in Supplementary Table 2.
X-ray examination. Radiographic examination was performed with a Faxitron cabinet X-ray system

RNA-seq analysis.
Total RNA-seq was performed as described previously [43][44][45] . In situ hybridization. In situ hybridization using 35 S labeling of an RNA-probe was performed as described previously 47 . The hybridization signals were imaged using a red filter, and the images were then superimposed on blue fluorescent images of the cell nuclei stained with Hoechst 33258 dye (Sigma-Aldrich). Additional information on the reagents used has been provided in Supplementary Table 1 Chondrocytes treated with recombinant protein and inhibitors. Primary chondrocytes were seeded on 24-well culture plates (6 × 10 4 cells/well) for RNA extraction or on 6-well culture plates (3 × 10 5 cells/ well) for protein extraction and incubated overnight. The cells were incubated in a fresh serum-free medium with recombinant proteins and/or inhibitors. The reagents used in this experiment are listed in Supplementary  Table 1. For the control group, the cells were treated with the same volume of the vehicle.
Three-dimensional microgravity cell culture and two-dimensional fast-rotating culture. The MG condition was generated using an RPM device (Yamato Scientific). The chondrocytes were seeded (3.5 × 10 5 cells/flask) on T25 flasks (Corning) and cultured overnight. The flasks were filled with culture medium without air bubbles and fixed on the RPM, positioned in an incubator set at 37 °C and supplied with 5% CO 2 . A centrifugal force of 1 g (1G) was established for the control.
Western blotting. Protein was extracted from cells using RIPA buffer supplemented with a Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and centrifuged; the supernatant was collected. An equal amount of lysate (20 μL), containing 5 μg protein, was loaded and resolved on a 10% SDS polyacrylamide gel (FUJIFILM Wako). The proteins were transferred onto a nitrocellulose membrane (Merck Millipore). The membranes were blocked using 3% BSA in Tris-buffered saline with Tween 20 and incubated overnight with the primary antibody. Antibodies used in this experiment are listed in Supplementary Table 1. The membranes were then incubated with a secondary HRP-linked antibody and incubated with ImmunoStar Zeta (FUJIFILM Wako) for generating the immunoreactive signal. The bands were detected with ImageQuant™ LAS 4000 (GE Healthcare).

Statistical analysis.
All results are presented as means ± SD. The sample size was determined based on previous studies 27 and was not calculated based on statistical power. Because of the small sample size, we could not guarantee the normality and homogeneous variance of each variable. Therefore, a non-parametric method was used for all statistical analyses. All results are presented as the median and interquartile range (IQR). The data were evaluated using a Mann-Whitney U test and Kruskal-Wallis test using GraphPad PRISM software version 8.0 (GraphPad, Inc.). The statistical test used in each experiment is indicated in the corresponding figure. A twosided P-value less than 0.05 was considered to reflect statistically significant differences.

Data availability
The RNA-seq data have been deposited in the Gene Expression Omnibus database under the accession code GSE173637. All other data supporting the findings of this study are available within the article, its Supplementary information files, and from the corresponding author upon reasonable request.